regulation of gpcr signalling Flashcards
be able to write out the pathways n shit here e.g. in an essay
what are GPCRs
g protein coupled receptors
largest receptor family
can be tissue specific
polypeptide chain of 300-500 amino acids
GPCRs are the largest receptor family with ____ members
> 800
approximately 500 GPCRs are expressed in ____ tissues
olfactory - sense of smell
(example of tissue specific)
___ to ___% of drugs in the market target GPCRs
30 - 50%
what are GPCRs characterised by (4)
7 transmembrane domains of alpha helices - largest family of membrane spanning receptors
distinctive structure
extracellular N terminus
intracellular C terminus
what shape does the transmembrane of a GCPR form and what does it contain
barrel - contains ligand binding site for ligand to bind to and activate
what are G proteins
heterotrimeric proteins of alpha, beta, gamma subunits
what does ligand binding in GPCRs cause
conformation change, leading to receptor acting as a guanine nucleotide exchange factor (GEF)
what is the role of GTP in GPCRs
GTP activates the G protein and causes dissasociation of the Ga G alpha subunit from the beta gamma subunit
what is the role of guanine nucleotide exchange factor GEF in GPCRs
loads G protein with GTP, which activates it
there are distinct Ga subunits that have different signal transduction through second messagers. true or false?
true
what is the signal transduction pathway of G alpha s (Gs) / stimulatory G alpha subunit
positive effect on adenylate cyclase
inc cyclic AMP (cAMP)
inc activation protein kinase A (PKA)
what is the signal transduction pathway of G alpha i (Gi) / stimulatory G alpha subunit
negative effect on adenylate cyclase
dec cyclic AMP (cAMP)
dec protein kinase A (PKA)
what is the signal transduction pathway of G alpha 12/13 (G12/13) / stimulatory G alpha subunit
activates Rac/Rho protein (a GTPase protein)
what is the signal transduction pathway of G alpha q (Gq) / stimulatory G alpha subunit
interacts with PLC phospholipase C which
converts PIP2= Phosphatidylinositol 4,5-bisphosphate
to IP3 = inositoltriphosphate and
DAG = diacylglycerol
IP3 inc PKC = Protein kinase C
inc Calcium ions leading to
activates CamKII = calmoduline-dependent kinase II
what do second messengers do
amplify signal
ie 1 mol activates GPCR, activates 1000 mols, downstream effects to 100,000 mol
e.g. Gi
GPCR signalling is not only determined by agonist binding, but is also ____ ____ by interactions with other receptors
allosterically modulated
some GCPRs dimerise when activated - what are these called and what do they mean (2)
homodimer - same GCPR
heteromers - different GCPR
type of dimer will impact GPCR signalling downstream
what are bivalent ligands
2 bound ligands that activate both GPCR heteromers
(each binds to GPCR, then form dimer, then other binds for third different effect)
aka form new ligand, only activates certain receptors if they are bivalent (stuck together in heterodimer)
receptor dimerisation is common in other types of receptors such as ___ ___ but remains controversial in the GPCR
tyrosine kinases
we are beginning to generate ligands that can activate both receptors in a dimer pair e.g. bivalent ligands
risk of cell signalling if not controlled
pathogenic signalling causing disease tumourogenisis
example of effect of pathogenic signalling
tumourogenisis, cancers
how to avoid pathogenic signalling
changing sensitivity of receptors can be adjusted based on levels of stimulation
(GPCR desensitisation)
changing sensitivity of receptors can be adjusted based on levels of stimulation. what is an example of this
rhodopsin is a GPCR in the rods of the retina - in very bright and dark light, sensitivity of receptors change - this is GPCR re/desensitising
what is GPCR desensitisation
waning/ reduction of cellular response in the face of continued or repeated stimulation - e.g. light
ie agonist response decreases
also conserves ATP
what is homologous desensitisation
the receptor only becomes desensitised to a specific agonist and normal response returns when a different agonist is used
what is heterologous desensitisation
receptor becomes desensitised to any agonist
how does activation of GPCRs reduce GPCR activation at a later time (8)
add agonist, activate GPCR
GPCR activation activates second messenger systems (e.g. cAMP) including PKA protein kinase
kinases phosphorylate proteins e.g. the activated GPCR when the G protein heteromer is dissociated (or coupled to G protein)
the newly phosphorylated sites on the GPCR cannot reassociate the G protein heteromer
this causes uncoupling of G protein from GPCR
the second messenger no longer promotes signal transduction from the receptor as ligand is less bound to GPCR
beta arrestin is recruited to replace G protein heteromer, blocks G protein heteromer blocking cell signalling
what do protein kinases do
phosphorylate proteins
example of a protein kinase
PKA
what does beta arrestin do (2)
block G protein heteromer by replacing it and binding to GPCR once it dissociates
inhibits downstream signalling transduction when interacts with GCPR aka signalling desensitisation
how do we know beta arrestin replaces G protein heteromer
we genetically engineer and attach green fluorescent protein (GFP) to beta arrestin
affect on beta-arrestin when receptor agonists e.g. isoproterenol is administered
forms clusters at cell membrane to bind to GPCR- moves away from defuse localisation translocates to membrane
shown as fluoresence
receptor agonist example
isoproterenol
the structure of beta arrestin suggests it is able to interact with proteins such as ___ and ___
clathrin
AP2
what are clathrin and AP2 involved in
endocytosis of receptors from cell membrane into cell to be altered and resensitised
means over time the receptor will gain sensitivity
how does clathrin and AP2 work
endocytic
these proteins bind and remove receptor from membrane
membrane invaginates
receptor eventually removed from EC facing environment
invagination eventually forms a vesicle containing GCPR - no longer on membrane
clathrin coated vesicle
eventually uncoats and forms early endosome
receptors dephosphorylate and recycle back to cell membrane to signal anew
GPCR no longer phosphorylated so G protein can recouple and GPCR is resensitised, transduction can occur
what is clathrin (4)
structural molecule
complex of light chain and heavy chain/ 2 proteins
3 legged structure/ trimeric
self form in cells as a lattice of hexagones, allows to bend membrane/ internalisations
what other outcome can occur for an early endosome (5):
downregulation:
GCPR as early endosome sent for degradation and broken down
results less receptors avaliable for signalling
receptor downregulation
long term, is why patients/addicts can present with drug tolerance - less receptors so inc dose needed
receptor signalling cannot recover when agonist present until new synthesis of receptor occurs
what is early endosome degradation caused by
lysosome
how does a degraded early endosome recover
receptor signalling cannot recover when agonist present until new synthesis of receptor occurs via transcription/ translation - cannot reuse
agonist should be removed also
beta arrestin is also a ____ ____
signal transducer - arrestin mediated signalling
what can beta arrestin activate
sarcoma (Src)
by accumulating clathrin coated vesicles
what is sarcoma (Src) used for
activates MAPK MAPkinase signalling pathway - cell growth, proliferation
what is the MAPK pathway also known as
Ras-Raf-MEK-ERK pathway
(each protein phosphorylates protein that comes after)
in the MAPK pathway, what is the function of ERK
translocates to nucleus and trigger transcription and translation
what, other than beta arrestin, can activate the MAPK pathway aka is a signal transducing protein unit
G protein beta gamma subunit
exact mechanism varies depending on cell type - can activate through Src or directly Ras
what can activate the PI-3 Kinase (PI3K) pathway
G protein beta gamma subunit
what does the PI-3 Kinase (PI3K) pathway activate
PKB/Akt
what does PKB/Akt do
inhibit apoptotic processes, contributing to cellular survival pathways
relevant to cancer – upregulated in cancer
what can activate ionotropic channels on cell membrane e.g. calcium ions from EC environment to IC environment or potassium ions from IC to EC
beta gamma subunit
what is an ionotrophic channel
undergoes conformational change when activated and allow ions to flow through
two ways GPCR signalling originates from
1- G protein signalling response mediated from alpha subunit and beta-gamma subunits
2- beta-arrestin signalling response, can produce distinct pathways not present in G alpha signalling pathways (1st option)
what is biased GPCR signalling
aka signalling bias
biased agonists can activate one signalling pathway over another (G protein signalling vs beta arresting signalling response)
example of biased agonist
oliceridine - opioid med
what does oliceridine act as an agonist for and what does it activate
biased agonist for mu opioid receptor
preferentially activates G protein signalling over beta arrestin signalling
effect of oliceridine in terms of drug tolerance
reduced beta arrestin recruitment
less receptor internalisation
reduced likelihood of drug tolerance as drug does not go through desensitisation or downregulation
common with drugs e.g. morphine that bind to mu opioid receptors
what do GCPRs transduce
signal from wide range of extracellular ligands
examples of extracellular ligands that GCPRs transduce
light, calcium ions, odorants, small molecules, proteins
is the GCPR N terminus intra or extracellular
extracellular
is the GCPR C terminus intra or extra cellular
intracellular
how are GCPRs activated
ligand binds
what is downstream signalling
also known as signal transduction pathways, are the processes that control how cells respond to signals and drive their overall activity
what is the most pharmacologically relevant heterotrimeric protein of G proteins
alpha (Ga) is the most pharmacologically relevant
what is GEF
guanine nucleotide exchange factor (GEF)
how is GTP activated
GEF guanine nucleotide exchange factor
displaces GDP and loads on GTP
the recruitment of beta arrestin requires
phosphorylation from PKA
if PKA null cell, no beta arrestin aka no clusters, diffused expression
what is resensitisation
recovery of cell responsiveness via endocytosis after desensitisation
then, can be resensitised
through dephosphorylation through protein phosphates like PP2A
and recycled back to cell membrane
When an RTK is activated it will form a dimer with another RTK. It will then ___________ the dimer partner to allow for adaptor proteins to bind.
transphosphorylate
Activation of PI3K by RTKs results in downstream signaling which activates which protein?
PKB
Receptor downregulation can be overcome by ______________.
Removing agonist and allowing new synthesis
class A GPCRs
interact with ligands in the EC
class B GPCRs
interact with peptides
class C GPCRs
are dimers
subunit G ____ allows for drug receptor complex specificity
alpha
as it is variable
what occurs in the intracellular C terminus when the GPCR binds
undergoes conformational change causing high affinity to the G protein
what anchors the GPCR
the beta gamma subunit
how else can amplification occur
freed active receptors can bind to other G proteins
cholera toxin
activates Gs singalling pathway via ADP-ribosylation of Gas subunit
dissociates Gas subunit from beta gamma subunit
activates adenylyl cyclase
inc cAMP excessively
inc PKA
phosphorylates/ opens ions channels in intestinal epithelium
inc flow of ions into intestine lumen
osmosis into lumen
profuse diarrhoea/ dehydration
aka cholera
carvediol
beta-adrenergic receptor blocker
signalling bias for beta-arrestin mediated signalling
clinically used for treating chronic heart failure
what is the difference between desensitisation and downregulation
desensitisation is a temporary reduction while downregulation is a long term decrease
desensitisation prevents overstimulation due to excessive signalling and loss of energy
downregulation leads to inc tolerance
what is a GRK
G-protein-coupled receptor kinase
phosphorylate activated GPCRs, marking for desensitisation
what two factors contribute to desensitisation
GRKs - account for some reduced signalling as leads to reduced interaction with G-protein
β-arrestin recruitment - accounts for most of desensitisation as outcompetes G-proteins
how were β-arrestins discovered
it was seen that β-arrestins prevented coupling of phosphorylated rhodopsin to the G-protein transducin
proved that β-arrestins do not readily dissociate, but G-proteins do
β-arrestin mutations
β-arrestins can be mutated to engage only one receptor site, leaving the other free to bind with the G protein, resulting in simoultaneous interaction
what type of desensitisation does salbutamol cause
homologous
B2 adrenergic receptor post exposure to salbutamol agonist
where do clathrin and AP2 bind
C termini
β-Adrenergic Agonists (e.g. albuterol) and dopamine agonists (e.g. fenoldopam) use which G pathway, and what effects result
stimulate G alpha
albuterol - inc cAMP leads to inc bronchodilation, good for asthma treatment
fenoldopam - inc cAMP, inc vasodilation, dec blood pressure
Morphine uses which G pathway, and what effects result - check pls lol
stimulates G inhibitory
dec cAMP
dec neuronal activity
analgesic effect
Currently, there are limited therapeutic agents that specifically target the ___ pathway. However, research is ongoing to develop modulators that can influence this pathway for potential therapeutic benefits.
G 12/13
